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The DNA binding domain (DBD) of poly(ADP-ribose) polymerase (PARP) has proved to be a novel, highly sensitive probe for detecting DNA breaks in intact cells undergoing apoptosis. A recombinant peptide spanning the DNA binding domain of PARP was expressed, purified and used to detect DNA strand breaks in fixed cells. Fluorescence microscopy with this probe followed by detection with anti-PARP antisera initially revealed an increased binding following treatment of cells with DNA strand-breaking agents (such asN-methyl-N'-nitro-N-nitrosoguanidine) and, subsequently, using biotinylated PARP DBD, during the later stages of apoptosis in several cell systems, when internucleosomal strand breaks became evident. This procedure was found to be at least as sensitive and required fewer steps to detect DNA strand breaks than those utilizing Klenow incorporation of biotinylated nucleotides.

We have investigated the effect of DNA damage on the expression of BCL-X, a member of the BCL-2 family. BCL-X mRNA levels were found to increase upon exposure human cells to ionizing radiation (IR). The Bcl-X(L) protein, but not Bcl-X(S), was identified to be induced by IR. Like BAX, another member of the BCL-2 family and a p53-regulated gene, the induction of BCL-X(L) was dependent on normal p53 function and required that cells have an apoptosis-susceptible phenotype. The induction of BCL-X(L) was rapid, transient and dose-dependent. The mRNA level peaked at 4 h and returned to baseline by 24 h post-irradiation. In agreement with the increased transcript level, Bcl-X(L) protein level was also observed to increase in cells with wild-type p53 where IR triggered apoptosis. In addition, a survey of the BCL-X(L) mRNA basal levels in human cells with known apoptotic responses showed that low basal levels of BCL-X(L) mRNA in cells were highly correlated with a strong ability of cells to undergo IR-induced apoptosis. On the other hand, high levels of basal BCL-X(L) were correlated with the resistance of cells to IR-induced apoptosis regardless of p53 status. These results indicate that BCL-2 and BCL-X(L) behave differently in response to DNA damage treatment even though they both are able to protect cells from p53-mediated apoptosis; along with down-regulation of BCL-2, BCL-X(L) was up-regulated by IR in human cells with wild-type p53 and susceptibility to IR-induced apoptosis. We speculate that the physiological function of increased BCL-X(L) protein would be expected to probably limit the severity and length of BAX effect in order to maintain a proper threshold for apoptosis and to complete cell cycle arrest activated by p53.

A fusion protein comprising the DNA-binding region of the glucocorticoid receptor and the catalytic domain of poly(ADP-ribose) polymerase was constructed. This chimeric protein was expressed both in E. coli and in eukaryotic cells and was recognized by antibodies to both polymerase and the glucocorticoid receptor. Similar to polymerase, the chimera produced bona fide poly (ADP-ribose) polymers covalently bound to protein and was inhibited by 3-aminobenzamide. Like the authentic glucocorticoid receptor, the fusion protein formed a stable complex with DNA containing the glucocorticoid response element. In mammalian cells, the fusion protein significantly and specifically inhibited the ability of the glucocorticoid receptor to stimulate a reporter construct. These results indicate that polymerase activity can be targeted to specific DNA sequences and modulate gene expression.

With an in vitro DNA repair system, Satoh and Lindah [(1992) Nature 356, 356-358] demonstrated that unmodified poly(ADP-ribose) polymerase (PADPRP) binds to radiation-damaged DNA and inhibits repair in the absence of NAD. However, in the presence of NAD, PADPRP undergoes automodification and the DNA is repaired. It was hypothesized that PADPRP cycles between an unmodified form, which protects DNA breaks, and an automodified form, which is released from DNA, thereby allowing access to repair enzymes. We have now tested this model with bacterially expressed mutants of PADPRP with deletions in the three major functional domains of the enzyme [Cherney, B. W., Chaudry, B., Bhatia, K., Butt, T. R., & Smulson, M. E. (1991) Biochemistry 30, 10420-10427]. Deletion mutants with an intact amino-terminal DNA-binding domain, and therefore capable of binding to DNA strand breaks in the in vitro assay, inhibited repair; however, whether the deletion was in the NAD-binding, active site domain or the automodification domain, the inhibition of repair exerted by these mutant proteins was not alleviated by NAD. A PADPRP mutant with a deletion in the DNA-binding domain did not inhibit DNA repair. Thus, the behavior of these PADPRP deletion mutants is consistent with the model proposed earlier. The model was also supported by experiments with Manley extracts of HeLa cells stably transfected with a PADPRP antisense RNA construct. Extracts of cells induced to express antisense RNA did not markedly inhibit in vitro DNA repair, nor did the addition of NAD influence the assay. In contrast, noninduced cell extracts inhibited repair and inhibition was alleviated by NAD.(ABSTRACT TRUNCATED AT 250 WORDS)

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.

We have determined the nucleotide sequence at the junction of the large single copy (LSC) region and the right and left members of the large inverted repeat, IRA and IRB, respectively, of the petunia chloroplast (cp) genome. As in Nicotiana debneyi and spinach (Zurawski et al. 1984), coding sequences of rps19 of petunia overlap the junction of IRB and LSC. Immediately into the LSC region upstream of IRA in the petunia cp genome are two small insertions relative to N. debneyi that occur at sites just inside IRA of N. debneyi. We discuss how these additions in one copy of the large inverted repeat of an N. debneyi-like ancestor to petunia resulted in shortening of the petunia large inverted repeat by 8 bases and in the resultant slight movement of rps19 farther into LSC. On a larger scale, the large inverted repeat in the tobacco, N. debneyi and petunia lineage relative to a spinach-like ancestor may have sustained several contractions due to deletions between short direct repeats found within IRA and the IRA/LSC junction. We also show how the large inverted repeat of N. debneyi instead may have been expanded relative to a tobacco-like ancestor by insertion into the large inverted repeat of bases between short inverted repeat sequences in LSC and the LSC/IRB junction.

TrnH and the intergenic region between trnH and psbA of the chloroplast genomes of alfalfa (Medicago sativa), Fabaceae, and petunia (Petunia hybrida), Solanaceae, were sequenced and compared to published sequences of that region from other members of those families. A striking feature of these comparisons is the occurrence of insertions/deletions between short, nearly perfect AT-rich direct repeats. The directionality of these mutations in the petunia, tobacco and Nicotiana debneyi lineages within the Solanaceae cannot be discerned. However, we present several alternative hypotheses that are consistent with Goodspeed's 1954 evolutionary treatment of the genus Nicotiana and family Solanaceae. Within the Fabaceae, the major size differences in the intergenic region between alfalfa, pea and soybean are due to insertions/deletions between direct repeats. The alfalfa intergenic region has an inverted repeat stem-loop structure of 210 bases directly 5' to trnH. This structure is an insert relative to the liver-wort. Marchantia polymorpha. Portions of the insert are found also in pea and soybean as well as in published sequences from other dicots representing diverse orders: petunia, tobacco, N. debneyi (Scrophulariales), spinach (Caryophyllales), and Brassica napus (Capparales). Some of the regions of the insert that are missing in these plants appear to have resulted from deletions of sequences between different imperfect direct repeats within, or 5' to and within the insert. Other deletions are not flanked by repeated sequences. A short insert flanked by imperfect direct repeats in B. napus occurs just within the longer alfalfa insert suggesting that both alfalfa and B. napus have remnants of an even longer insert relative to M. polymorpha. From these analyses we hypothesize the insertion of a stem-loop structure into an M. polymorpha-like ancestral land plant, followed by deletions of sequences, often between different imperfect direct repeats within and upstream of the insert, leading to the psbA-trnH intergenic sequences represented by the present-day plants examined.